FIGURE 2. Tim4-mediated engulfment of apoptotic cells.

a, NIH3T3/DNase II (filled squares, open squares) and NIH3T3/DNase II/Tim4 (filled circles, open circles) incubated with 3.0  10⁵ (filled squares, filled circles) or 6.0  10⁵ (open squares, open circles) apoptotic CAD^-/- thymocytes for the indicated periods. The percentage of TUNEL⁺ macrophages was determined by flow cytometry. Average values from three experiments are shown; error bars, 1s.e. b, NIH3T3/DNase II and NIH3T3/DNase II/Tim4 used for 60-min phagocytosis, stained for TUNEL, and observed by microscopy. Arrows indicate TUNEL⁺ cells. c, NIH3T3/DNase II or NIH3T3/DNase II/Tim4 incubated with healthy or apoptotic thymocytes in the presence of Kat5-18, stained for TUNEL. The percentage of TUNEL⁺ macrophages was determined by flow cytometry. Average values from three experiments are shown; error bars, 1s.e.. d, CAD^-/- mice that had received normal hamster Ab or Kat5-18 were treated with dexamethasone. Top, the thymus stained for TUNEL (green) and F4/80 (red). The number of TUNEL⁺ cells determined for 50 randomly selected macrophages (bottom). Experiments were carried out three times, and the percentage of the macrophages carrying more than 15 apoptotic cells plotted (right). Mann–Whitney's U-test was used to analyse the difference, and P values are shown. e, Normal hamster IgG, Kat5-18 or PBS was injected into mice twice a week for 5 weeks. The serum concentration of anti-cardiolipin and anti-dsDNA was determined at 2, 5 and 10 weeks. P values determined as above are shown. A₄₉₂, absorbance at 492 nm.